Part:BBa_K2260002:Experience

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Applications of BBa_K2260002

An assay was conducted in order to measure the effect of our part, Phasin-HlyA, on PHB secretion. Triplicates of E. coli BL21 containing either Phasin-HlyA and PhaCAB or only PhaCAB (as a control) were placed in LB media containing 3% glucose, and allowed to grow for 48 hours. PhaCAB, the gene that produces PHB, ensured that equal amounts of PHB were produced in both our test and the control. The PhaCAB part came from Imperial College 2013, and can be found at https://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1149052.

At 24 hour intervals, live cells were separated from the media using differential centrifugation. This resulted in two fractions: a cellular fraction containing the live cells, and a secreted fraction containing the PHB that was released into the media, either through secretion or cell death. Before separation, CaCl₂ was added to agglomerate PHB particles together.

The fractions were then treated with with Triton X-100 in PBS to remove cellular debris, and washed to isolate PHB particles. The pellets were allowed to dry overnight, before being weighed and compared. Masses were then adjusted to account for the addition of CaCl₂, and the final masses were compared. Our results (seen below) showed that our part, Phasin-HlyA, led to a 114% increase in secreted PHB found in the media compared to a control cell without our secretion part.

Our results showed that our part, Phasin-HlyA, led to a 114% increase in secreted PHB found in the media compared to a control cell without our secretion part.

User Reviews

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